Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, contains five copies of the ESAT-6 gene cluster, each encoding a dedicated ESX protein secretion system which has been defined as a novel Type-VII secretion system. The ESX have been implicated in virulence and survival of M. tuberculosis, and as such present a promising target for novel treatment interventions. This study has investigated the evolution, regulation, functions and substrates of the ESX secretion systems. The evolutionary history of the ESX secretion systems was established using in silico and phylogenetic analyses of the sequenced mycobacteria, closely related actinomycetes and WXG-FtsK clusters from other bacteria. The ESX-4 gene cluster appears to have evolved with the start of the evolution of the mycomembrane, followed by the duplication of ESX-3, which marks the evolution of the genus Mycobacterium. The ESX-1 duplication occurred next, followed by ESX-2 and ESX-5 which occur only in the slow growing mycobacteria. Five additional ESX gene clusters were newly identified and named ESX-P1 to - P5. These additional ESX clusters occur, or are predicted to occur, on plasmid DNA, and appear to be progenitors of the genomic ESX-1 to -5 gene clusters, possibly indicating a plasmid-mediated mechanism of ESX duplication and evolution. The promoters expressing the M. tuberculosis ESX-1 to ESX-5 secretion systems were investigated using a promoter probe assay, and characterised using in silico analyses. Promoters were identified for ESX-1, -2, -3 and -5. The functions of the mycobacterial ESX secretion systems were investigated using whole proteomic, secretomic and metabolomic analyses of the fast growing, non-pathogenic M. smegmatis, which contains three of the ESX secretion systems, ESX-1, 3, and 4. ESX knockout strains of M. smegmatis were generated and used in comparative analyses with wild-type M. smegmatis. ESX-1 was highly expressed in wild-type M. smegmatis, however no specific pathways showed considerable variation when ESX-1 was deleted. Deletion of ESX-3 resulted in substantial variation to multiple cellular pathways, including amino acid, carbohydrate and fatty acid metabolism and oxidative stress. These and other differences indicate possible perturbed polyamine metabolism in the absence of ESX-3. Although no ESX-4 protein components were detected in wild type M. smegmatis, the ESX-4 knockout displayed substantial proteomic variation. Reduced levels of ESX-3 component proteins in the ESX-4 knockout suggest that ESX-4 influences ESX-3 expression. Other variation linked ESX-4 to cell division and molybdenum metabolism. Secretomic analyses of wild-type and ESX knockout M. smegmatis strains were used to search for novel substrates of the M. smegmatis ESX secretion systems. No prototype ESX substrates were identified in the culture filtrates, however 10 possible substrates of the ESX-1, -3 and -4 secretion systems, containing the general ESX secretion signal, YxxxD/E, were identified. The functions of some of these proteins correlate with the ESX functions identified in the proteomic and metabolomic analyses. This study sets the groundwork for future work in understanding the functional roles and expression patterns of these ESX secretion systems and in using global proteomic and metabolomic analyses to understand cellular changes in response to specific signals or genomic changes. / AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis, die veroorsakende agent van tuberkulose, bevat vyf kopieë van die ESAT-6 geengroep, wat elk 'n toegewyde ESX proteïen sekresiesisteem, omskryf as 'n nuwe Tipe-VII sekresiestelsel, kodeer. Die ESX sekresiesisteme is betrokke by virulensie en oorlewing van M. tuberculosis, en is dus belowende teikens vir nuwe behandelings. Hierdie studie het die evolusie, regulasie, funksies en substrate van die ESX sekresiesisteme ondersoek. Die evolusionêre geskiedenis van die ESX sekresiesisteme is bepaal met behulp van in silico en filogenetiese analises van die volgordebepaalde mikobakterieë, nouverwante actinomisete en WXG-FtsK groepe van ander bakterieë. Die ESX-4 geengroep het saam met die evolusie van die mikomembraan ontwikkel, gevolg deur die duplisering van ESX-3, wat die evolusie van die genus Mycobacterium merk. Die ESX-1 duplisering het volgende plaasgevind, gevolg deur ESX-2 en ESX-5, wat slegs in die stadiggroeiende mikobakterieë voorkom. Vyf addisionele ESX geengroepe is nuut geïdentifiseer in hierdie studie en is ESX-P1 tot -P5 genoem. Hierdie addisionale ESX groepe is op, of word voorspel om op, plasmied DNS voor te kom, en mag voorlopers van die genomiese ESX-1 tot -5 geengroepe wees, wat moontlik dui op 'n plasmied-gemedieërde meganisme van ESX duplisering en evolusie. Die promoters wat verantwoordelik is vir die uitdrukking van die M. tuberculosis ESX-1 tot ESX-5 sekresiesisteme is ondersoek deur middel van 'n promoter aktiwiteitstoets, en gekarakteriseer deur in silico analises. Promoters is geidentifiseer vir ESX-1, -2, -3 en -5. Die funksies van die mikobakteriële ESX sekresiesisteme is ondersoek deur proteomiese, sekretomiese en metabolomiese analises van die vinnig-groeiende, nie-patogeniese mikobakterium M. smegmatis, wat ESX- 1, -3 en -4 sekresiesisteme besit. ESX uitslaanmutante van M. smegmatis is gegenereer en gebruik in die vergelykende analises met die wilde-tipe M. smegmatis. ESX-1 is hoogs uitgedruk in wilde-tipe M. smegmatis, maar geen spesifieke metabolise weë het aansienlike variasie getoon wanneer ESX-1 verwyder is. Delesie van ESX-3 het gelei tot aansienlike variasie in verskeie sellulêre weë, insluitend aminosuur-, koolhidraat- en vetsuur-metabolisme en oksidatiewe stres. Hierdie en ander verskille dui op moontlike versteurde poli-amien metabolisme in die afwesigheid van ESX-3. Hoewel geen ESX-4 proteïenkomponente opgespoor is in wilde-tipe M. smegmatis nie, vertoon die ESX-4 uitslaanmutant aansienlike proteomiese variasie. Laer vlakke van ESX-3 proteïne dui daarop dat ESX-4 die uitdrukking van ESX-3 beinvloed. Baie van die proteomiese variasie kan geassosieer word met verlaagde ESX-3 uitdrukking, maar ander variasie mag ESX-4 koppel met seldeling en molibdeen metabolisme. Sekretomiese analises van wilde-tipe en ESX uitslaanmutant M. smegmatis stamme is gebruik om nuwe substrate van die M. smegmatis ESX sekresiesisteme te identifiseer. Geen prototipe ESX substrate is geïdentifiseer in die kultuurfiltraat, maar 10 moontlike substrate van die ESX-1, -3 en -4 sekresiesisteme, met die algemene ESX sekresiesein, YxxxD/E, is geïdentifiseer. Die funksies van sommige van hierdie proteïene korreleer met die funksies geïdentifiseer in die proteomiese en metabolomiese analises. Hierdie studie stel die grondslag vir toekomstige werk in die begrip van die funksionele rol en uitdrukkingspatrone van die ESX sekresiesisteme en in die gebruik van globale proteomiese en metabolomiese analises om sellulêre veranderinge in reaksie op spesifieke seine of genomiese veranderinge te verstaan. / The National Research Foundation / German Academic Exchange Service (DAAD), / The Harry Crossley Foundation / The Ernst and Ethel Erikson Trust / Stellenbosch University